Total RNA of cell lines was by TRIzol (15576425, Invitrogen, USA), and then cDNA was further synthesized by PrimeScriptTM RT reagent Kit (RR039A, TaKaRa, Japan). Pre-synthesized gene primers (Sangon, China), Roche SYBR Green Master (05629017212), and DEPC water were added to the cDNA and mixed together and tested in the detection instrument (thermal cycler T100, Bio-Rad, USA), according to the following settings: pre-denaturation at 95 °C for 10 minutes (min), denaturation at 95 °C for 15 s, annealing at 58 °C for 1min, for a total of 40 cycles. For calculating RNA levels, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were internal references. The primer's sequences were shown in Table 1 . The amplification efficiencies of BANCR, miRNA-590-5P, LC3-I, LC3-II, Beclin-1, P62, OLR1, GAPDH, and U6 were 97.2%, 94.6%, 103.7%, 101.3%, 95.0%, 99.5%, 104.7%, 96.0%, and 98.1%, respectively. The relative level was calculated by the 2-△Ct method.
Gene primers
Manufactured by Sangon
Sourced in China
Gene primers are short, single-stranded DNA sequences used in molecular biology and genetics to initiate the replication of a specific DNA fragment during the polymerase chain reaction (PCR) process. They serve as the starting point for DNA synthesis, allowing the targeted amplification of genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green mix, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Sybr green master mix, by Vazyme (1 mentions)
Cfx connect, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
4 protocols using gene primers
1
Quantitative Real-Time PCR Analysis
2
Renal Cell Carcinoma Gene Expression Analysis
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Twenty-four pairs of ccRCC tissue samples were obtained from the Department of Urology, Union Hospital, Tongji Medical College, Wuhan, China. The study was compliant with all relevant ethical regulations regarding research involving human participants. Total RNA was extracted from samples by the TRIzol reagent (Thermo, Massachusetts, USA) and then reverse-transcribed. The resulting cDNA was then subjected to real-time PCR analysis with SYBR Green mix (Thermo, Massachusetts, USA). Results were normalized to GAPDH for each sample. Gene primers are obtained from Sangon Biotech (Shanghai) and list as follows:
GAPDH
Forward 5′‐GAGTCAACGGATTTGGTCGT‐3′
Reverse 5′‐GACAAGCTTCCCGTTCTCAG‐3
AQP9
Forward 5′‐CCACCAGAAGACGATTAAGCCACAG‐3′
Reverse 5′‐GGTCCTCACTTTCACAGCCAGATG‐3′
IBSP
Forward 5′‐GCACCAGTACCAACAGCACAGAG‐3′
Reverse 5′‐GGCTCCAGTGACACTTTCTTCTTCC‐3′
STEAP3
Forward 5′‐CGGAGGTCATCTTTGTGGCTGTG‐3′
Reverse 5′‐GCTGAAGGTGCTCTTGCTCTGTAG‐3′
GAPDH
Forward 5′‐GAGTCAACGGATTTGGTCGT‐3′
Reverse 5′‐GACAAGCTTCCCGTTCTCAG‐3
AQP9
Forward 5′‐CCACCAGAAGACGATTAAGCCACAG‐3′
Reverse 5′‐GGTCCTCACTTTCACAGCCAGATG‐3′
IBSP
Forward 5′‐GCACCAGTACCAACAGCACAGAG‐3′
Reverse 5′‐GGCTCCAGTGACACTTTCTTCTTCC‐3′
STEAP3
Forward 5′‐CGGAGGTCATCTTTGTGGCTGTG‐3′
Reverse 5′‐GCTGAAGGTGCTCTTGCTCTGTAG‐3′
3
Cardiac Gene Expression Analysis
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Trizol (Invitrogen Co., Carlsbad, CA, United States) was used to extract RNAs from homogenized myocardia (n = 4 hearts/group). After the normalization of RNA concentrations, cDNAs were synthesized using Revertra ace® qPCR rt kit (Toyobo, Osaka, Japan). By using SYBR Green Master Mix (Vazyme Biotech, Nanjing, China), the following gene primers (Sangon Biotech, Shanghai, China) were used to evaluate mRNA expressions; (1) Tumor necrosis factor-alpha (TNF-α), Forward GAAAGCATGATCCGAGATGTG; Reverse: CACGAGCAGGAATGAGAAGAG, (2) transforming growth factor-beta (TGF-β1), Forward: ATGGTGGACCGCA ACAACGC; Reverse: CTGGCACTGCTTCCCGAATGTC, (3) inducible nitric oxide synthase (iNOS), Forward: TCTTGGAGCGAGTTGTGGATTGT; Reverse: TAGGTGAGG GCTTGCCTGAGTG, (4) arginase 1 (Arg-1), Forward: CGTTG ACCTTGTCTTGTTTTGG; Reverse: CTGGTTCTGTTCGGT TTGCTG, (5) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Forward: TCCTGCACCACCAACTGCTTAG; Reverse: AGTGGCAGTGATGGCATGGACT.
The 2–ΔΔCt analysis method was used to evaluate the relative mRNA levels as described (Gold et al., 2012 (link)) and have been graphically presented as fold changes compared with the Sham group.
The 2–ΔΔCt analysis method was used to evaluate the relative mRNA levels as described (Gold et al., 2012 (link)) and have been graphically presented as fold changes compared with the Sham group.
4
Rabbit Knee Cartilage mRNA Expression
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The medial femoral condylar cartilage of the rabbit’s right leg was taken, and total mRNA was extracted with Trizol (15596026, Ambion, America). Total mRNA was reversely transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, America). Finally, RT-qPCR was performed thrice using the CFX ConnectTM fluorescence quantitative PCR instrument with the PowerUpTM SYBRTM Green Master Mix (A25742, Thermo Scientific, America). The sequences of gene primers (Sangon Biotech Co., Ltd. Shanghai) used for measurement are shown in Table 1 . Glyceraldehyde 3-phosphate dehydrogenase mRNA was used as an endogenous control. Relative transcript levels of mRNA were reported using the 2−ΔΔCT method.
The gene primers
| Gene | F/R | Primers (5’ to 3’) |
|---|---|---|
| COL2A1 | Forward | CCCAGAACATCACCTACCA |
| Reverse | CAGTCTTGCCCCACTTACC | |
| MMP13 | Forward | TGCGGGAATCCTGAAGAAGAATGC |
| Reverse | TCAAGTTTGCCTGTCACCTCTAAGC | |
| ITG-β1 | Forward | GCCATCCAGACGACATAGAGAATCC |
| Reverse | TGCCTTTGCTACGGTTGGTGAC | |
| GAPDH | Forward | ACTCTGGCAAAGTGGATGTTGTCG |
| Reverse | CCGTGGGTGGAATCATACTGGAAC |
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